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原发性肝癌是一种高度恶性肿瘤,在引起肿瘤相关性死因中居第二位,大多数肝癌病人就诊时已处于进展阶段,无法接受根治性治疗,预后差,复发率和病死率高[1-2]。肝细胞癌(hepatocellular carcinoma, HCC)约占原发性肝癌中的90%[2]。欧洲肝脏病学会和美国肝脏病学会推荐的治疗HCC的主要治疗手段包括手术切除、经动脉化疗栓塞术、肝移植及局部消融等[3]。尽管近年来这些手段有了一定的发展,但由于肝癌转移和复发率较高,预后仍较差。所以,迫切需要寻找新的治疗方法。
近年来,伴随着免疫学、分子生物学、生物工程的发展,肿瘤免疫治疗已成为最有潜力的治疗手段之一。肿瘤中浸润的巨噬细胞称肿瘤相关巨噬细胞(tumor associated macrophages,TAM),为一类重要的免疫细胞。TAM被认为主要是M2型巨噬细胞[4],主要作用是免疫抑制,促进血管生成,分泌多种促进肿瘤生长的因子等。核因子κB(nuclear factor κB,NF-κB)是一种细胞核转录因子,广泛存在于真核细胞中。NF-κB参与了机体炎症反应、免疫应答及细胞凋亡等,可以通过调节细胞因子等,促进肿瘤血管生成、浸润和转移。NF-κB p50是其重要家庭成员之一,对于NF-κB蛋白在肿瘤中扮演的角色起到重要作用。但NF-κB与M2巨噬细胞的关系尚不明确。本文研究了肝癌中M2巨噬细胞及NF-κB p50的表达情况,探讨它们在不同分期肝癌中表达的相关性。
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绿色荧光标记CD206蛋白,红色荧光标记CD163蛋白,DAPI染细胞核,Merge可显示绿色和红色荧光共定位情况,两种荧光重合的细胞表示该细胞存在CD206和CD163的共定位。免疫荧光双染结果显示,对照组中CD163和CD206的共表达较少,且荧光强度较弱,而TNMⅠ+Ⅱ期病人肝组织切片中CD163和CD206的共表达增多,荧光强度增加。随着肝癌进展,TNM Ⅲ+Ⅳ期中CD163和CD206的共表达明显比TNMⅠ+Ⅱ期病人增多,且荧光强度明显增强(见图 1)。
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Western blotting结果显示,CD163和CD206在肝癌病人肝组织中表达上升, Ⅲ+Ⅳ期病人上升较Ⅰ+Ⅱ期病人更为明显(P<0.05~P<0.01)(见图 2、表 1)。
分组 CD163 CD206 p50 对照组 0.32±0.05 0.34±0.05 0.31±0.04 Ⅰ+Ⅱ期 0.48±0.07** 0.62±0.04* 0.85±0.04** Ⅲ+Ⅳ期 0.81±0.08**△△ 0.88±0.07**△△ 1.79±0.04**△△ F 40.044 66.482 991.034 P <0.01 <0.01 <0.01 MS组内 0.005 0.003 0.003 注:相对灰度值=目的蛋白灰度值/β-actin灰度值; q检验:与control组比较*P<0.05, **P<0.01;与Ⅰ+Ⅱ期组比较△△P<0.01 表 1 不同分期肝癌病人新鲜肝脏组织中CD163、CD206、p50表达相对灰度值的比较(x±s; ni=3)
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Western blotting结果显示,NF-κB p50的表达在肝癌病人肝组织中上升, Ⅲ+Ⅳ期病人上升较Ⅰ+Ⅱ期病人更为明显(P<0.01)(见图 3、表 1)。
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相关性分析发现,肝癌病人肝脏组织中NF-κB p50与CD163、CD206的表达在各组中呈现明显正相关(r=0.963, P<0.01;r=0.944, P<0.01)(见图 4)。
肝细胞癌中M2巨噬细胞标志物与NF-κB p50相关性研究
Expression of M2 macrophages in hepatocellular carcinoma, and its correlation with NF-κB p50
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摘要:
目的探讨不同分期肝细胞癌病人组织中M2巨噬细胞及核因子κB(NF-κB)p50的表达情况,并分析其表达的相关性。 方法整群收集2012-2015年80例肝细胞肝癌病人和10例肝内胆管结石病人(对照组)的组织标本,采用免疫荧光双染法检测CD163和CD206的表达;收集2015年10月至2016年1月20例接受肝细胞癌手术病人的新鲜组织标本,采用Western blotting法检测不同分期的肝癌组织中CD163、CD206的表达及NF-κB p50的表达。 结果肝癌病人石蜡组织切片中CD163和CD206的共定位高于对照组,Ⅲ+Ⅳ期肝癌切片中的共定位高于Ⅰ+Ⅱ期;Western blotting结果表明,肝癌组织中CD163、CD206及NF-κB p50的表达高于癌旁组织(P < 0.05~P < 0.01),Ⅲ+Ⅳ期肝癌组织中的表达高于Ⅰ+Ⅱ期(P < 0.01)。NF-κB p50与CD163、CD206的表达均呈正相关(r=0.963、0.944,P < 0.01)。 结论随着肝癌进展,M2巨噬细胞数量和NF-κB p50的表达均升高,提示两者可能具有协同作用,共同促进肝癌进展。 Abstract:ObjectiveTo evaluate the expression of M2 macrophages and nuclear factor κB(NF-κB) p50 in patients with different stages of hepatocellular carcinoma(HCC), and analyze the relationship between them. MethodsThe expression levels of CD163 and CD206 in 80 cases of paraffin sections of patients with HCC and 10 cases of patients with hepatolithiasis(control group) were detected.The expression levels of CD163, CD206 and NF-κB p50 in 20 cases of fresh liver tissues of patients with different stages of HCC were detected using Western blotting from October 2015 to January 2016. ResultsThe co-localization of CD163 and CD206 in paraffin section of HCC was higher than that in control group, and the co-localization of which in TNM Ⅲ+Ⅳ stage HCC was higher than that in TNM Ⅰ+Ⅱstage HCC.The results of Western blotting showed that the expression levels of CD163, CD206 and NF-κB p50 in hepatocellular carcinoma tissue were higher than those in para-carcinoma tissue(P < 0.05 to P < 0.01), and the expression levels of CD163, CD206 and NF-κB p50 in Ⅲ+Ⅳ stage hepatocellular carcinoma tissue were higher than those in Ⅰ+Ⅱ stage hepatocellular carcinoma tissue(P < 0.05 to P < 0.01).The expression of NF-κB p50 was positively correlated with the expressions of CD163 and CD206(r=0.963, r=0.944, P < 0.01). ConclusionsWith the development of liver tissue, the number of M2 macrophages and NF-κB p50 level increase, which suggest that the two may have synergistic effects to promote the progression of liver cancer. -
表 1 不同分期肝癌病人新鲜肝脏组织中CD163、CD206、p50表达相对灰度值的比较(x±s; ni=3)
分组 CD163 CD206 p50 对照组 0.32±0.05 0.34±0.05 0.31±0.04 Ⅰ+Ⅱ期 0.48±0.07** 0.62±0.04* 0.85±0.04** Ⅲ+Ⅳ期 0.81±0.08**△△ 0.88±0.07**△△ 1.79±0.04**△△ F 40.044 66.482 991.034 P <0.01 <0.01 <0.01 MS组内 0.005 0.003 0.003 注:相对灰度值=目的蛋白灰度值/β-actin灰度值; q检验:与control组比较*P<0.05, **P<0.01;与Ⅰ+Ⅱ期组比较△△P<0.01 -
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